This invention relates to methods of screening for compounds capable of inducing apoptosis in certain tumor-cells. The invention also relates to compounds identified by such methods. In addition, the invention relates to methods for the in vitro diagnosis of Xeroderma pigmentosum and compounds useful in these methods.
Certain tumors, benign, premalignant, and malignant, are known to have genetic components etiologically. The gene for the nuclear phosphoprotein, p53, is the most commonly mutated gene identified in human cancers. Missense mutations occur in tumors of the colon, lung, breast, ovary, bladder, and several other organs. When mutant forms of the p53 gene are introduced into primary fibroblasts, these cells are immortalized. The wild type p53 gene can suppress the growth of transformed human cells, but oncogenic forms lose this suppressor function. Thus, the p53 gene has been termed a xe2x80x9ctumor suppressorxe2x80x9d gene.
If the p53 gene of a tumor cell is of the wild type, its p53 gene product may nevertheless be interfered with functionally. For example, a transforming viral infection of the cell can interfere with the p53 protein product. For instance, certain strains of human papillomavirus (HPV) are transforming and are known to interfere with the p53 protein function because the virus produces a protein, E6, which promotes degradation of the p53 protein.
There is also pharmaceutical interest in p53 because p53 protein is capable of inducing certain tumor cells to undergo apoptosis. In apoptosis, or xe2x80x9cprogrammed cell deathxe2x80x9d, a series of lethal events for the cell appear to be generated directly as a result of transcription of cellular DNA. Thus, apoptosis is a physiologic means for cell death. For example, lymphocytes exposed to glucocorticoids die by apoptosis. Involution of hormone sensitive tissue such as breast and prostate that occurs when the trophic hormone is removed occurs via apoptosis.
In particular, recent studies have indicated that the introduction of wild type (non-mutated) p53 into transformed cell lines that carry a mutant form of p53 induces the cells to undergo apoptosis with disintegration of nuclear DNA. It is believed that p53 may suppress tumor development by inducing apoptosis, thus modulating cell growth.
In addition to p53, there are numerous other genes involved with cell growth. One group of such genes is designated XP because their derangement can result in the disease Xeroderma pigmentosum. Xeroderma pigmentosum is a rare disorder characterized by disfigurement, deranged pigmentation of the skin, scarring and heightened incidence of skin cancers, especially on exposure to sunlight. The disease is inherited as an autosomal recessive trait. Eight genetic forms of the disease are known. Phenotypically these forms vary in their symptoms, signs and severity. Two of the more grave forms are associated with mental deficiencies. These two forms are characterized by mutations in the XPB and XPD genes.
Selection of drugs for potential therapeutic use against tumors is an area of medical research which remains fraught with complications and which often present an array of suboptimal treatment choices. There are currently a multitude of potential compounds available to evaluate. Screening procedures are valuable to limit the bewildering array of drug choices for further testing. Improvements in screening methods or reagents are highly desirable. In addition, there is a need for better diagnosis of XP subtypes. These and other needs are addressed by the present invention.
The invention provides a method for screening a compound for an ability to induce apoptosis. The method includes providing a first cell containing either a normal or mutant p53 gene. The first cell is responsive to p53. For instance, the first cell is typically capable of undergoing apoptosis after microinjection of a DNA construct expressing wild type p53. The method further includes providing a second cell containing at least one mutant Xeroderma pigmentosum gene such as a mutant XPB gene, or a mutant XPD gene, or both. The second cell is not usually capable of undergoing apoptosis after microinjection of a DNA construct expressing wild type p53. According to a method of the invention, both the first and second cells are contacted with a compound of interest. An example of a compound of interest is any compound one desires to screen for possible use as a chemotherapeutic agent or drug. The method includes detecting whether or not apoptosis of either the first or second cell, or both, occurs after contact of the compound to the cells. A comparison of the observations for apoptosis is made, thereby determining whether the compound can induce apoptosis.
The first and the second cell can be selected from any of a number of cell types including benign, premalignant, and malignant. The first and the is second cell can be uninfected or infected. If the latter, the infection can be viral, such as from a papilloma virus. The first and second cell can be selected from any histological or anatomical classification. Typically, the cells are selected from the group consisting of fibroblastic, epithelial, and hematopoietic cells. The cells can be derived from a variety of tissues, including tissue of colon, lung, breast, ovary, cervix, liver, kidney, nervous system, and hematopoietic system. Preferably, the cells are fibroblastic or lymphoblastic cells.
The invention also provides a method of screening for a compound capable of inhibiting the binding of p53 protein to a Xeroderma pigmentosum protein, such as either XPB or XPD proteins or both. This method includes providing a reagent having at least one Xeroderma pigmentosum protein, preferably XPB or XPD, or both. The reagent is contacted with the compound, permitting the compound to compete with wild type p53 protein for a binding site on any or all of the Xeroderma pigmentosum protein(s). Subsequently, any binding of the compound to the protein(s) is detected.
Additionally, this method can include contacting the reagent with wild type p53 protein and detecting a binding of the wild type p53 to at least one of the Xeroderma pigmentosum proteins such as an XPB and or XPD protein(s). The method can further comprise attaching a label to at least one of the Xeroderma pigmentosum protein(s) and the p53 protein. The label can be any of a number of detectable labels known in the art. Some examples are an antibody, a radioisotope, and a fluorescent molecule. Conveniently, the reagent has a TFIIH complex containing both XPB and XPD proteins.
The invention also provides a method of screening for a compound capable of inhibiting at least one Xeroderma pigmentosum helicase activity, such as XPB and or XPD helicase activity. This method includes providing a reagent having at least one Xeroderma pigmentosum protein, contacting the reagent with the compound which permits thee compound to bind to the Xeroderma pigmentosum helicase, and determining the helicase activity. Typically, the reagent has a TFIIH complex containing both XPB and XPD proteins.
The invention also provides compositions. In particular this invention describes compounds consisting essentially of an amino acid sequence selected from a group of subsequences from wild type p53 having the 393 amino acids depicted in SEQ ID NO:1 wherein the subsequences are in their native order and selected from the group consisting of: (a) amino acids 319 to 393 (SEQ ID NO:7); (b) amino acids 361 to 393 (SEQ ID NO:8); (c) amino acids 367 to 387 (SEQ ID NO:2); (d) amino acids 350 to 380 (SEQ ID NO:9); (e) amino acids 355 to 375 (SEQ ID NO:10); (f) amino acids 360 to 370 (SEQ ID NO:11); and, (g) a subsequence comprising a, b, c, d, e or f where the subsequence further consists of amino acids of SEQ ID NO:1 which flank subsequence a, b, c, d, e, or f within 10 amino acids or less at either or both of the amino or carboxy terminus and are in their native order with the proviso that the peptide have less than 100 amino acids, and wherein said compound: (1) binds to a binding site on at least one of the XPB helicase and the XPD helicase; (2) competes with wild type p53 proteins for the binding site; and, (3) inhibits the helicase activity. Preferred compounds include the above compounds wherein the compound is a peptide consisting of subsequence d, e, or f wherein the subsequence is flanked at either or both the amino and carboxy terminus by amino acids of SEQ ID NO:1 in their native order that naturally flank and are 10 amino acids or less from the subsequence d, e or f with the proviso that the compound has less than 80 or 50 amino acids.
Unless otherwise stated all numerical references are inclusive of the numbers set forth.
A compound of the invention can be used in a diagnostic method, such as a method of diagnosing a Xeroderma pigmentosum complementation group, preferably group B or D, in an individual. Such a method includes providing a sample cell derived from the individual, contacting the sample cell with a compound of the invention, and detecting whether or not apoptosis of the sample cell occurs, thereby diagnosing whether or not the sample cell contains at least one mutant Xeroderma pigmentosum gene, preferably a mutant XPB gene, a mutant XPD gene, or both.
Another compound of the invention consists essentially of the amino acid sequence depicted in SEQ ID NO:4 wherein the compound possesses at least one, and typically two, of the following properties: (1) it binds to a binding site on wild type p53 protein and (2) it competitively inhibits the binding of wild type p53 protein to wild type XPB protein. Preferably, this compound consists of the amino acid sequence depicted in SEQ ID NO:4. Another method of diagnosing a Xeroderma pigmentosum complementation group, such as group B or D, in an individual includes providing a sample cell derived from the individual, contacting the sample cell with a compound of the invention, and detecting whether or not apoptosis of the sample cell occurs, thereby diagnosing whether or not the sample cell contains at least one mutant Xeroderma pigmentosum gene such as a mutant XPB gene, a mutant XPD gene, or both.
Finally this invention provides for methods of inducing apoptosis in a cell having lost its native p53 regulatory control of apoptosis said method comprising the administration to said cell of an effective amount of a peptide derived from the carboxy terminus of p53 to induce apoptosis wherein the peptide consisting essentially of an amino acid sequence selected from a group of subsequences from p53 having the 393 amino acids depicted in SEQ ID NO:1 wherein the subsequences are in their native order, have two (an amino and carboxy) termini, and are selected from the group consisting of: (a) amino acids 319 to 393 (SEQ ID NO:7); (b) amino acids 361 to 393 (SEQ ID NO:8); (c) amino acids 367 to 387 (SEQ ID NO:2); (d) amino acids 350 to 380 (SEQ ID NO:9); (e) amino acids 355 to 375 (SEQ ID NO:10); (f) amino acids 360 to 370 (SEQ ID NO:11); and, (g) a subsequence comprising a, b, c, d, e or f wherein the subsequence is flanked at either or both the amino and carboxy terminus by amino acids of SEQ ID NO:1 which naturally flank subsequence a, b, c, d, e, or f within 10 residues or less and are in their native order with the proviso that the peptide have less than 100 amino acids, and wherein said compound: (1) binds to a binding site on at least one of the XPB helicase and the XPD helicase; (2) competes with wild type p53 proteins for the binding site; and (3) inhibits the helicase activity. The preferred method uses peptides where the peptides comprise subsequence d, e, or f wherein the subsequence consists of amino acids of SEQ ID NO:1 in their native order which are 10 residues or less from the subsequence with the proviso that the peptide be less than 80 or 50 amino acids. In some embodiments the peptide will be fused or coupled to a second peptide capable of facilitating the translocation of the peptide across the nuclear membrane of a cell.